Accepted Manuscript Methanobrevibacter smithii and Bacteroides thetaiotaomicron cultivated from a chronic paravertebral muscle abscess Vanessa Demonfort Nkamga, Romain Lotte, Pierre-Marie Roger, Michel Drancourt, Professor Raymond Ruimy PII:
S1198-743X(16)30394-9
DOI:
10.1016/j.cmi.2016.09.007
Reference:
CMI 719
To appear in:
Clinical Microbiology and Infection
Received Date: 8 August 2016 Revised Date:
14 September 2016
Accepted Date: 14 September 2016
Please cite this article as: Nkamga VD, Lotte R, Roger P-M, Drancourt M, Ruimy R, Methanobrevibacter smithii and Bacteroides thetaiotaomicron cultivated from a chronic paravertebral muscle abscess, Clinical Microbiology and Infection (2016), doi: 10.1016/j.cmi.2016.09.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Methanobrevibacter smithii and Bacteroides thetaiotaomicron cultivated from a chronic
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paravertebral muscle abscess
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Vanessa Demonfort Nkamga1, #, Romain Lotte2, 3, 4, #, Pierre-Marie Roger5, Michel
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Drancourt1, and Raymond Ruimy2, 3, 4,*
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Unité de recherche sur les maladies infectieuses tropicales et émergentes, Marseille, France
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Laboratoire de bactériologie, Centre Hospitalier Universitaire de Nice, Nice, France
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Université de Nice Côte d’azur, Faculté de Médecine, Nice, France
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Inserm U1065, C3M, Team6, “Bacterial toxins in host pathogen interactions”, Bâtiment
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universitaire Archimed, Nice, France.
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Nice, France
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*Corresponding author: Professor Raymond Ruimy
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Laboratoire de Bactériologie
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Centre Hospitalier Universitaire de Nice, Hôpital de l'Archet II,
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151 route de Saint Antoine de Ginestière CS 23079 06202 Nice Cedex 3, France
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Tel: 33 (0) 4 92 03 62 14
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E-mail:
[email protected]
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Keys words: Abscess, Archaea, Bacteroides, Methanobrevibacter, Metronidazole,
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Service de maladies infectieuses et tropicales, Centre Hospitalier Universitaire de Nice,
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equally contributing authors
Fax: 33 (0) 4 92 03 59 52
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ACCEPTED MANUSCRIPT Sir,
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Methanobrevibacter smithii is the main human-associated methanogenic archaea being part of
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the vaginal, oral and gut microbiota. As a methanogen, it is capable of detoxifying molecular
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hydrogen resulting from fermentation by anaerobes communities, into gaseous methane. To
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date, M. smithii has been associated in human with weight gain and obesity and with colonic
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diseases including ulcerative colitis, Crohn’s disease and colorectal cancer [1-3]. However, its
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role in other human diseases remains controversial and might be underestimated because of
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difficulties associated with its culture and detection. Indeed, it requires a strict anaerobic
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atmosphere comprising up to 80% hydrogen to grow and it can be detected by specific
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amplification and sequencing which are not routinely used in clinical laboratories.
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Applying specific archae 16S rRNA [4] and methyl coenzyme M reductase (mcrA)
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gene [5] amplification on ten human internal abscesses from various origins, we detected M.
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smithii in a paravertebral muscular abscess from a 41-year-old man. In July 2014, the patient
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visited the emergency department of our tertiary care center for night sweats, chronic fever
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and worsening of a lumbar round-shaped tumefaction over the previous three months.
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Physical examination showed a right-sided and painless paravertebral tumefaction. The lesion
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showed mild signs of inflammation and no cutaneous fistula was observed. Pelvic
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examination showed a right lymphadenopathy of the inguinal area with no evidence of
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tenderness or inflammation symptoms. There appeared to be no cutaneous, abdominal,
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neurological, and cardio-vascular abnormalities upon physical examinations. Body
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temperature was 39°C. Blood tests revealed elevated inflammation marker values including
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C-reactive protein (75.9 mg/L) and leukocytosis (11.4 x109/L). A full-body computed
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tomography scan and magnetic resonance imaging revealed an abscess of the right transverse
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muscle, and the diagnosis of chronic abscess of the transverse muscle was highly suspected,
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but a neoplastic process was not excluded. Surgical drainage of the lesion was performed and
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ACCEPTED MANUSCRIPT drainage fluids and biopsies were sent to the laboratory for pathological, bacteriological
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(including slow growth bacteria and mycobacterium) and mycological analyses. An antibiotic
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treatment consisting of amoxicillin and clavulanic acid was initiated intravenously 2 g three
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times a day (t.i.d) for seven days, followed by oral metronidazole 500 mg t.i.d for three
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weeks. Histopathological findings after surgery confirmed the chronic non-specific
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granulomatous lesion with a polymorphic inflammatory infiltrate and excluded a neoplastic
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process. Direct microscopic examination of the pus revealed Gram-negative rods and
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leukocytes. Culture on blood agar remained sterile under aerobic atmosphere and grew
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Bacteroides thetaiotaomicron identified by MALDI-TOF mass spectrometry (Bruker
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Daltonics, Germany) under anaerobic atmosphere. This anaerobic Gram-negative bacillus is a
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major component of the physiological gut flora and is an opportunistic pathogen that can be
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involved in severe infection such as septicemia or abscesses formation from multiple body
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part [6]. To further investigate the presence of methanogenic archaea, the sample was
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cultivated in SAB-medium as previously described [7]. After three-week incubation, methane
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production was detected in tube culture. Sequencing of archaea 16S rRNA and mcrA gene
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PCR products yielded a sequence similarity (accession number: LT223565 and LT223564) of
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99% with the reference M. smithii ATCC 35061 homologous sequences (Figure 1). We tested
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the susceptibility of M. smithii paravertebral strain against metronidazole and amoxicillin +
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clavulanic acid, by transferring 500 µL of an exponentially growing culture into 4.5 mL of
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fresh SAB-medium as previously described [8]. Briefly, two tubes of culture medium were
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supplemented to obtain the following antibiotic concentration: metronidazole (B. Braun
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Medical SAS, Boulogne-Billancourt, France) 1µg/mL and amoxicillin + clavulanic acid (8:1;
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BIOGARAN, Colombes, France) 100 µg/mL. The tubes were incubated at 37°C under
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agitation and growth of archaea was observed after 3 days of incubation. Control cultures
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were also incubated without antibiotics to provide a baseline for growth of archaea. The
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ACCEPTED MANUSCRIPT growth was monitored by measuring methane in the tubes using gas chromatography (Clarus
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500, Perkin Elmer, Courtaboeuf, France). The isolate was resistant to amoxicillin + clavulanic
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acid (MIC value ≥100µg/mL), and was susceptible to metronidazole (MIC value ≤1µg/mL).
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The data reported herein was in accordance with the previous knowledge about the
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antimicrobial susceptibility patterns of human methanogens [8]. Clinical and biological
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follow-up at three week confirmed the complete healing of the patient.
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We isolated for the first time M. smithii in a paravertebral muscle abscess using specific
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culture and NA-based approach for methanogens. Interestingly, B. thetaiotaomicron was
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previously described as the major source of hydrogen needed for methanogenic archaea
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metabolism in a humanized gnotobiotic mouse model [9]. Our study suggests that M. smithii,
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previously known as a commensal organism, could play a role in human infections and
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notably in chronic abscesses in association with anaerobic bacteria such as B.
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thetaiotaomicron. We can therefore hypothesize a metabolic cooperation between these two
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organisms. M. smithii is naturally resistant to beta-lactams which are the most used empirical
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antibiotics to treat patients presenting with deep abscesses. Fortunately, in most cases, the
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empirical antibiotic treatment also takes into account the presence of anaerobic bacteria and
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includes metronidazole which is also active against methanogenic archaea. Further studies are
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still required to precise the epidemiology of archaea in human infections and the potential
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occurrence of resistant strains.
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Transparency declaration: All authors report no conflicts of interest for this article.
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Acknowledgment: This work has been supported by a Grant of “Mediterranean Infection
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Institute” (Marseille, France) to Vanessa Demonfort Nkamga, and by a grant of FRM
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(Grant#FDM20-150632804) to Romain Lotte. 4
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References
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[8]. Khelaifia S, Brunel JM, Raoult D, Drancourt M: Hydrophobicity of imidazole derivatives
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Samuel BS, Gordon JI: A humanized gnotobiotic mouse model of host-archaeal-bacterial mutualism. Proc Natl Acad Sci U S A 2006, 103: 10011-10016.
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ACCEPTED MANUSCRIPT Figure 1 legend: Unrooted neighbor-joining tree based on 16S rRNA sequences showing the
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phylogenetic position of Methanobrevibacter smithii LT223565 (paravertebral isolates)
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within members of genera Methanobrevibacter. The tree was performed using MEGA6
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software as previously described. Values above lines are bootstrap values expressed as
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percentages (only values>90% are shown). Scale bar indicates accumulated changes per
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nucleotide.
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Figure 1.
Methanobrevibacter smithii AY196669 Methanobrevibacter smithii LT223565
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Methanobrevibacter smithii U55233
Methanobrevibacter oralis HE654003 Methanobrevibacter millerae AY196673
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Methanobrevibacter gottschalkii U55238
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Methanobrevibacter olleyae AY615201 Methanobrevibacter ruminantium AY196666
Methanobrevibacter arboriphilus AY196665
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Haloferax alexandrinus NR 113438
